The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

1.It is difficult to understand and analyse the figures when the descriptions and text are not exact, or sometimes impossible to read.Authors are strongly encouraged to proofread the manuscript and correct inaccuracies -Label of fig1A is missing, is the second panel M2-STOP228-EGFP?Yes, the reviewer is correct.We had inadvertently brought the panel on top of the label, and this mistake has now been corrected.We apologies for this error.We do not see cells in the insert (brightfield) of fig1A of non transfected.We have enhanced the contrast of the inset of Fig1A in order to better visualize the cells, that are present albeit they do not display any signal in the GFP-channel.-Fig2 is difficult to understand Annotations are of very bad quality and impossible to read -Fig2A and 2C text is not of good quality, it is very difficult to read the text.Idem for Fig3, the text is not visible Concerning these well founded comments by the reviewer, we overall apologise sincerely because there was clearly an issue in the PDF conversion.Albeit a source word file had been provided, including high-resolution figures, clearly the low quality of the manuscript hampered the work of the reviewer.We made sure that figure quality is now preserved in the resubmission.The minor labelling issues raised by the reviewer have been thoroughly addressed.
-Lane 240 "This is as also confirmed from ribosome profiling data from human left ventricle tissue, displaying increased p-sites as well as overall ribosomal coverage in correspondence of this region (Fig. S4B)".what are "p-sites" ? Figure S2B is just impossible to read Ribosome profiling is a method that uses specialized mRNA sequencing to determine which mRNAs are being actively translated in a cell or tissue.It produces a global snapshot of all the ribosomes actively translating in a cell at a particular moment, known as a translatome.Consequently, this enables the identification and the location of translation start sites (P-site), the complement of translated ORFs in a cell or tissue, the distribution of ribosomes on a messenger RNA, and the speed of translating ribosomes.The P-site (for peptidyl) is the second binding site for tRNA in the ribosome.The other two sites are the A-site (aminoacyl), which is the first binding site in the ribosome, and the E-site (exit), the third.During protein translation, the P-site holds the tRNA which is linked to the growing polypeptide chain.An increase in P-site coverage is normally associated to translation initiation.
-Graph Fig4B: impossible to read the Y axis legend Again, this has been fixed after PDF conversion was improved.-FigS8 legends: S8A "below" is not the lower panel but the right panel We thank the reviewer for pointing this out, we have corrected this.
-Fig S10B-S10D legends are too small-Impossible to read Again, this has been fixed after PDF conversion was improved.

Controls are needed
2.1.Line 122-FigS2A-Table1: To fully validate the hypothesis of a reconstitution of competent M2 receptor with the Nter and Cter parts of the recepror when a stop codon is introduced at position 228 of M2, coexpression of the Nter fragment (1-228, comprising only TM1-5 trunk, without the rest in the DNA construct) with the Cter tail ) would be necessary.Indeed the residual binding observed with M2-Stop228 could be due to expression of a weak fraction of full length M2 despite the introduction of a stop at position 228 since ribosomes can have the properties to go through and pass STOP codon.Same remark for M3 in Table S2 and figS5.Furthermore, Western Blot profiling of the different constructs should also be shown -We thank the reviewer very much for these comments.Indeed, we have already performed the N-ter + C-ter co-expression experiment, which was not included in the current manuscript version to streamline the flow.By constructing the mutants M3stop273/fr.sh.and M3stop273/Hairpin, we have also ruled-out stop-codon readthrough for the M3stop273 receptor (see Table S3).

The table below, also included in
We shall note here that, especially for GPCRs, we believe western blots are not the most suitable tool for validating these constructs, and that radioligand binding data combined with direct, single-cell microscopic imaging are more sensitive and reliable approaches.However, according to the recommendation from the reviewer and in order to prove that M2stop228 produces an N-terminal fragment that ends at amino acid 228 we conducted a western blot where we run side to N-terminally  3).Detection was conducted using 1:1000 anti myc-Tag (9B11) mouse primary antibody (Cell Signaling).After overnight incubation, the membranes were washed 3x 10 minutes in TBS-T and incubated at RT for 1 h with a secondary antibody (1:10000 anti-mouse IgG/(anti rabbit) HRP-linked antibody from Cell Signaling).Below, loading control using beta actin.Blue bars indicate the relative ratio of C terminal domain band intensity to full length receptor band.
show an increase of the expression of the C-terminal fragment for increasing times of starvation.

Inaccuracies and concerns:
-Fig3B: M2 is not well visible at the cell surface in 1B in contrast to what is seen in other figures We thank the reviewer for this comment.In this specific case, the receptor is indeed expressed at the membrane.However, we focused the imaging plane on to the basal membrane, i.e. the one adhering to the coverslip.In line to previous reports, this leads to a diffuse homogeneous signal unlike the more common 'annular' intensity profile that is observed when the imaging plane crosses the cell 'halfawy' through its axial extent.We enclose here a library of images of the basolateral membrane of the M2.For comparison, we include here several confocal snapshots of the basal membrane of HEK293 cells expressing several different fluorescently labeled GPCRs.-Fig3E: mitochondrial localization is not possible to see clearly, the images are not convincing.In addition, separate spectral green channel displayed at the bottom does not seem to correspond to the one from the merge top image.
We are sorry that the reviewer does not find the image as convincing.We would like to respectfully point out that reviewer 2 instead appears convinced by the 'large' degree of colocalization of green signal in mitochondria.We felt the folded mitochondria at the center of the cell were a good example of mitochondrial localization.We do agree, however, that there are also some green particles that do not colocalize with mitochondria.This is a common instance with labeled proteins, that may be degraded, or trafficked in vesicles.In any case, we include here for the -Lane 340 and FigS7: The authors wrote "Similar mitochondrial localization was also observed when we transfected HEK293 cells with M2fr.sh-mRuby2 (Fig. S7B)."However in FigS7B, mitotracker staining is missing.We thank the reviewer for this observation.We have altogether repeated the measurement, and now figure S7B displays 4 panels, clockwise for M2tail(368-466)-EGFP, M2fr.sh-mRuby2 (in red), overlay and DIC.In the top right cell the colocalization is perfect.Please not that the lower left cell expressed the M2fr.sh-mRuby2 but no M2tail(368-466)-EGFP.
-Fig4A images are of very poor quality -impossible to judge the integration into mitochondria We apologize deeply once again for the image conversion problem.The images at the appropriate resolution are now included.
-FigS8: FigS8: scales are indicated to be 10um; however the scale bar size is different in all images but cell size seem to be similar….
There is no nuclear staining here, for some images, it would be necessary to include it We thank the reviewer for these comments and suggestions.In Figure S8 the scale bars wereindeed all 10 µm, and when a larger field of view was chosen (as in the case of the former Figure S8E, this was done to show that the negative control did not show any signal across a larger population of cells.In any case, following the suggestion by the reviewer, we have incorporated several more images (there are now 40 individual micrographs) of our fluorescence complementation controls, including large field of view micrographs of the negative controls, and including DIC for all images (which we believe, provides equivalent information to that conveyed by a nuclear staining, i.e. allows to identify also non-transfected cells).We hope the information provided is now acceptable to the standards of the reviewer.
Idem nuclear staining of S6 would help.Plasma membrane is difficult to distinguish We imagine the reviewer is referring here to Figure S6   Review figure 8 independent transfections of M2tail(368-466)-GFP11 and SMAC-GFP1-10 in HEK293 cells (as also shown in Figure S8E).Scale bar is 10 µm throughout.
-Lane 442 and Fig5C-5D: "The green fluorescence detected at the plasma membrane is explained with the chaperoning effect of M2-GFP11 on Mito-GFP1-10 before its sorting to the mitochondria".This observation calls into question the Mito-GFP1-10 sensor.Indeed Mito-GFP1-10 being observed at the plasma membrane instead of mitochondria suggests that the mitochondrial sensor is not specific; What are the comments of the authors regarding this aspect ?
We thank the reviewer for raising this valid and insightful comment.The localization of two complemented proteins when the GFP11-GFP1-10 pair is used will, in our experience, be determined by which protein takes the lead in terms of subcellular localization.In Figure 5C we believe that cap dependent receptor production is dominant, and when the receptor is expressed and targeted to the membrane it carries with it any mito-GFP1-10 that it finds around in the cytosol.On the other hand, when the C-terminal fragment is upregulated under stress and it is targeted to the mitochondria we observe a predominant mitochondrial signal.The latter can arise both from a recombination with mito-GFP1-10 prelocalized in the mitochondria (due to the 24 hours headstart in transfection of this construct) and from the chaperoning effect of the M2tail(368-466)-GFP11 on any mito-GFP1-10 being concomitantly transcribed.
-The physiological role of Ctail is not clear, its impact on cellular apoptosis or proliferation is not clear.Indeed: FigS10D: Expression of M2stop228, M2Stop400 and M2tail increases cleaved caspase 3 compared to WT, suggesting that expression of the 3 constructs including C-tail induced some cell death (while WT does not).However, the authors wrote in the text Lane 483: "no difference in the number of apoptotic cells among the different cell groups as shown by the caspase-3 activation assays of apoptosis" Is there a discrepancy here ?Does the Ctail increase apoptosis ?
We thank the reviewer for this comment.We agree with the reviewer that we are only beginning to understand the potential physiological roles of Ctail (i.e.M2tail(368-466 or M2 C-terminal fragment).However, based on our data, we can say with confidence that in absence of its IRES-mediated, physiological expression, cell proliferation and oxygen comsumption rates are increased.This occurs both when the M2Receptor is overexpressed, but also, importantly, at endogenous expression levels, as demonstrated by our data from Crispr/Cas9 edited hiPSCs reported in Figure 7C-E.
Concerning the Caspase assay of Figure S10D: we are afraid that our choice of showing the overexposed blot in the bottom part of the figure has caused some confusion.The key result is that there is no sizable cleaved Caspase 3 for any of the lanes observed.The amount of cleavage, when looking at the overexposed blot, is in line with that of the control construct M2 stop400.In this construct no c-terminal is made, hence any Cleaved Caspase 3 band represents the 'background noise'.Moreover, of note, the already faint M2tail band is fainter than the negative control M2 stop400 band.We include here, for the reviewer's benefit, an example of HEK293 cells where Caspase cleavage instead occurs upon Taurosporine treatment of the cells.The blot shows that under untreated conditions there is a faint band of cleaved Caspase 3, but this is negligible with respect to the intensity of the band when cleavage is induced by Taurosporine.
Ctail-induced apoptosis would also match the increased cell viability assay on HEK293 cells transfected with M2-M368A (ie in cells not expressing Ctail) (Fig6), or the cell proliferation assay performed on M368A hiPSCs (Fig7D), compared to WT receptor Extra experiments would certainly be needed to clarify this aspect In order to address this concern, in Figure S10E we show that the mitochondrial network of cells overexpressing (for 24-48h) M2tail(368-466)-EGFP is in excellent condition, and this does not change upon serum starvation.We hope we have clarified this concern and doubt of the reviewer.

Minor:
Lane 230: Is the word "However" appropriate here ?
We thank the reviewer for this and the subsequent careful proofreading comments.
We have omitted the "however".Lane 240, "This is as also confirmed… » remove "as" done Lane 328 A word is missing at the end of the sentence: " ….the mechanism of C-terminal fragment production is therefore close to physiological." We have changed the sentence by adding "close to physiological expression".
Fig3D legends, remove « as well as strongly to the cell mitochondria »; since nothing in 3D show that the localisation is in mitochondria; replace by intracellular compartments or organelles.Idem in the text lane 332 We have amended the caption in Figure 3D.However, we believe that in the case of Figure 3E we indeed show mitochondrial localization, as testified by the counterstain.We feel have addressed this question thoroughly in response to the major point above raised by the reviewer.
-Fig2 "OF" is missing in the Title of fig2: "Localization of the IRES within the i3 loop OF the muscarinic M 209 2 receptor using a fluorescence reporter assay." We thank the reviewer for spotting this typo, that is now amended.
The different constructs of fig2A are not easy to understand; adding colors for the different parts of the i3 loop fragments would help the reader We thank the reviewer for this suggestion, and we have indeed coded the different sections.We have also improved the graphics of the barcharts displayed in Figure 2A and B Fig4F-G: specify in the legend the antibody associated with gold particles, and also needs controls with cells without M2 We thank the reviewer for pointing this out.The secondary, gold conjugated antibody used is EM Grade 10nm Goat anti-Mouse IgG Conjugate EM.GAM10/1, from BBC UK, and is now clearly spelled in the Methods section.We shall note here that the primary antibody was an anti myc-Tag (9B11) mouse primary antibody (Cell Signaling), and so we obtained Gold particle counts only in transfected cells.Furthermore, COS-7 cells were transfected with M2stop228-myc not M2-myc.The M2-myc would have produced two recognizable proteins, the full length M2-myc receptor and the M2tail-myc fragment, while the M2stop228-myc produces only one recognizable protein that is the M2tail-myc fragment that has a predominant mitochondrial localization.The results of our counts in transfected vs non-transfected (without M2stop228myc) cells is displayed below, together with a representative comparison of a control (immunostained, from untransfected cells, LEFT) and a transfected and immunostained (right) IEM micrographs.We shall note here that the background granularity observed in the negative control has spatially broader and less defined features than those of the sharp gold nanoparticles observed in the positive staining, as can be seen when enlarging the images.
Review figure 10 Statistics of IEM staining, both in transfected as well as control cells.Representative IEM micrographs of a control (left) and transfected (right) COS-7 cells.Scale bar is 1 µm.
-Figure S9 FigS9D colocalisation should be dispalyed in white In this specific figure we used blue instead of magenta to render mitotracker signal, as magenta would have been too close to red.We shall note that blue and red gives purple/magenta, as colocalization color.-Figure S10 Put a distinguishable box to differentiate the top and bottom panels of S10D We thank very much the reviewer for this sensible suggestion, that we have duly implemented.
Put a box to distinguish the 3 panels of S10E We thank the reviewer for this observation.We have spaced the panels slightly in order to distinguish them

Rev. 2:
In this paper, the authors reported that there is an internal ribosome entry site (IRES) in the third intracellular loop of M2 receptor which splits M2 receptor into two fragments.They showed that the carboxyl terminal fragment locates to the mitochondria inner membrane and regulates various functions including oxygen consumption and cell proliferation.The authors employed various techniques and a lot of different constructs to comprehensively map out the IRES site, report the subcellular localization and assess the functions.I think this is interesting and potentially impactful, but I have a few comments as detailed below.
We thank the reviewer for this fair assessment.We shall note here however that the IRES does not actually split the receptor in two, but yields, on top of the canonical, full length M2 receptor, also a C-terminal fragment (M2tail) starting from the end of the third intracellular loop.So the full length receptor and the C-terminal fragment are both present.This concept is explained in the scheme of Fig. 8 1.How often does cap-independent translation of M2 happen and does it have significant impact in physiological conditions?Majority of M2 receptor locates at the plasma membrane as shown by the authors themselves and many other papers.If less than 5% locates to the mitochondria, would that have any significant impact?
We thank the reviewe for pointing this out, and also for supporting the notion that indeed there is a strong colocalization with mitotracker.The dots that do not colocalize could be C-terminal fragment being degraded into vesicles, or being trafficked.We shall remember that the C-terminal fragment retains two transmembrane domains, so will tend to 'live' in membranes, either mitochondrial or endosomal.In our work we have used both HEK293 and COS7 cells.COS7 cells were used for most of the radioligand binding experiments, whereas HEK cells were primarily used for microscopy.However, for consistency and verification, most of the microscopic imaging and biochemistry was conducted on both cell types as for example reported in Figure S6J (current version) (formerly Figure S7C).We shall further note here that we tested IRES activity in three further cell types, as displayed in Figure S4A, and more recently also in H9c2 cells (as shown in the reply to Reviewer 1 above).

In
In Figure 1A, the top right panel illustrates a COS7 cell, the other panels are HEK293.The confusion about 4 vs five panels arises because the sentence was supposed to be at the end of the caption and refer to all imaging panels.We have now corrected this.
Page 15, line 418, over a 4h interval?Do you mean 1h interval?
What we mean here is that after transfection, we waited 5h and then started imaging for an additional 4h (over a 4h interval).Images were on the other hand taken every 30 minutes, as shown in Figure 4B.

ReviewerFigure 4
tagged myc-M2stop228 myc-M2trunk(1-228), I.e. the construct where the DNA sequence is interrupted after coding for aa 228.The data clearly show that the Nterminal portion of M2stop228 and M2trunk have comparable molecular weights (~55 kDa, accounting for likely glycosilation).Reviewer Figure1Western blot of whole-cell lysates of untransfected HEK293 cells (Lane 1, N.C.= negative control), HEK293 transfected with myc-M2Stop228 (Lane 2) and myc-M2Trunk(1-228) (Lane 3).Detection was conducted using 1:1000 anti myc-Tag (9B11) mouse primary antibody (Cell Signaling).After overnight incubation, thes were washed 3x 10 minutes in TBS-T and incubated at RT for 1 h with a secondary antibody,1:3000 antimouse IgG HRP-linked antibody from Cell Signaling).On the lower part is shown a loading control, αTubulin (11H10) rabbit primary antibody (1:1000) detect with a secondary antibody (1:3000) antirabbit IgG HRP-linked from Cell Signaling) Reviewer Figure2Western blot of whole-cell lysates of , HEK293 transfected with M2-myc (Lane 1) M2Tail(1-368)-myc (Lane 2) and untransfected HEK293 cells (Lane 3, N.C.= negative control).Detection was conducted using 1:1000 anti myc-Tag (9B11) mouse primary antibody (Cell Signaling).After overnight incubation, the membranes were washed 3x 10 minutes in TBS-T and incubated at RT for 1 h with a secondary antibody (1:10000 anti-mouse IgG/(anti rabbit) HRP-linked antibody from Cell Signaling).On the lower part is shown a loading control, αTubulin (11H10) rabbit primary antibody (1:1000) detect with a secondary antibody (1:3000) anti-rabbit IgG HRP-linked from Cell Signaling) For comparison, the full length receptor runs in several bands, the highest of which at around ~75 kDa (as also shown in Figure S6K), and prominently dispaying the C-terminal associated fragment bands running as a doublet at 15 and 21 kDa, that are eventually localized to the mitochondria, as shown by the blot in Figure 3C.As indicated in the manuscript, we ascribe the higher molecular weight band of the two arising from posttranslational modifications.The offset between the 15 kDa tail band originating from the full length receptor and that from the C-terminal fragment itself, is in agreement with the effect arising from the slightly different linker used to fuse the myc tag.In addition to this, and to further characterise our constructs, as requested by the reviewer, we performed western blot with the M2stop228-Myc mutants expressed in COS7 cells.The blot shows that the M2stop228 runs as two bands, with the lower molecular weight band matching the M2tail(368-466), and the upper molecular weight band corresponding to the 1-228 trunk portion of the receptor.Review figure 3 WB M2/C-Myc, M2stop228/C-Myc, M2stop400/C-Myc and M2tail(368-466)/C-Myc containing the human Myc tag (EQKLISEEDL) at Cterminal were transiently transfected in COS-7 cells and immunodetected via Western Blot after SDS-Triton protein extraction.Both M2 wild type, M2stop228 and M2tail(368-466)/C-Myc showed 15 kDa bands corresponding to the segment of the M2 receptor from ATG 368 up to the C-terminal.The 75 kDa bands indicated by the arrows in M2 WT corresponds to the full lenght wild type M2 receptor.On the contrary, M2stop400 did not show any band representing an internal control.TableS1: Explain why the coexpression of MT2-STOP196 + M2-Trunk1-283 shows some binding; properties it is rather unexpected.Furthermore, controls of expression of all the constructs must be provided; without checking level of expression of mutant constructs, conclusions can be erroneous.Same remarks for the experiments related to the M3 receptor The M2-stop196 mutant is an additional control that we provided to demonstrate that the stop codon must be inserted within the i3 loop in order to split the receptor in a functional way.Insertion of the stop codon in transmembrane regions, generate fragments that are unable to bind ligands.In the M2-stop196 mutant, the stop codon in the middle of Transmembrane region V provides a truncated fragment that is unable to reassemble with the tail part.Nevertheless, the M2tail fragment generated by the IRES mediated mechanism, can reassemble with a cotransfected M2-Trunk1-283 and regain [3H]NMS binding.The same reasoning can be done for the mutant M3stop240 cotransfected with M3trunk, since again the stop codon is in transmembrane region V.2.3 Fig3C:The authors should show the expression of each construct in the total lysate compared to the mitochondrial fraction.Evaluation of expression via IRES vs cap dependent expression would be interesting to know.We thank the reviewer for this comment.Indeed, we had already provided this result in the supplementary findings, see Fig.S6K.We have now added a legend to guide the reader as to the origin of the different bands observed in the blot in the total lysate.Prior to our experiments reported in Figure5, we had conducted in the past an experiment where me measured the relative increase (blue bars) of the Cterminal fragment with respect to the full length receptor in HEK293 subject to increasing starvation, a driver of increased stress response and thus of IRES activity (Reviewer Figure4).The data qualitatively Western blot of whole-cell lysates of , HEK293 transfected with M2-myc and lysed upon increasing times of starvation (medium without serum).( 2.4.Fig 4C in vitro import into yeast mitochondria: I have the feeling that some controls are lacking: for ex, same condition of expression and import but this time in the absence of yeast mitochondria ?We believe that the best control in this type of experiment is the full length M2 receptor protein that does not enter in the mitochondria (left portion of the blot).It is important to stress the fact that in this assay the M2 and M2tail(368-466) proteins, are synthetized in vitro in presence of a 35S radiolabelled methionine and after purification they are added to the mitochondrial preparation.The first line (5%), in both left and right blots of Fig. 4C, represents the aliquot of the M2 (left) or M2tail(368-466) (right) synthetized in vitro, and added to the mitochondrial preparation.
B-F.In this case, as the focal plane is at the plasma membrane, any nuclear staining would be very faintly visible, if at all.For this reason, we believe that, by using a colorscale that emphasizes low intensities in panel C and F, we have actually done the best possible effort to highlight the plasma membrane.In the other panels of FigureS6, we are only showing colocalization of two different proteins/stains at the level of the mitochondrial network.

Fig S8A :
Fig S8A: Indicate the MW of M2 Ctail incorporated into yeast mitochondria?Is it the one <17kDa We thank the reviewer for this comment, since the molecular weights were indeed not indicated in this panel and have now been added.Indeed, what wasd isplayed in Supplementary Figure 8A (now Fig. 7C in the revised version) is the 15 kDa M2tail fragment.

FigS9H à
FigS9H à mitochondria (mitotracker) are barely visible Again scale of the images on top and bottom are not the same We thank the reviewer for these observations.Figure S9 has been re-edited, and we introduced in panel D magnified images of mitochondrial localization of IRES generated M2tail(368-466)-mRuby2 or absence thereof in panel I. Scale bars are always 10 µm, but in this case the two original images were taken with slightly different pixel numbers (albeit same pixel size), and we refrained from cropping panels.
Figure4 E, F, G.I could see many similar particles in the images.Are these not real signals?Please clarify.We thank the reviewer for checking carefully our electron microscopy images.While at low zoom the gold nanoparticles are hard to identify and distinguish from overall 'noise' and other sources of contrast, at higher magnifications we feel their identification (H-K)is unambiguous.We shall note furthermore that the Regions of Interest in Figure4Fand Figure 4G are arbitrary, and do not include all particles visible in the mitochondria.For Example, there are other gold nanoparticles visible in Figre 4F (to the top and left of the H square). Minor: Fig. 1A.top right panel add text M2stop228-EGFP.We thank the reviewer for pointing out that the text was masked by the Figure.Fig. 1A.Are they HEK293 or COS7 cells?"Imaging setting are the same for the five images" five or four?there are only four images in figure 1A.

codon readthrough could not account for the weak observed binding in M2stop228, since in Table S1 we showed that the constructs, M2stop228/fr.sh., in which, a four nucleotides segment to shift the reading frame was inserted after the stop codon 228, was still able to bind specifically [3H]NMS. Another construct, shown in Table S1, M2stop228/Hairpin, in which an hairpin loop was inserted after the stop codon 228, excludes stop-codon readthrough. Hairpin loops are stable structures that have been shown to block ribosome scanning (Kozak M. Constraints on re-initiation of translation in mammals. Nucleic Acids Res. 2001;29: 5226-5232. doi:10.1093/nar/29.24.5226; Kozak M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989;9: 5134-5142. doi:10.1128/mcb.9.11.5134-5142.1989), but despite the the hairpin loop, M2stop228/Hairpin still binds [3H]NMS.
Review table 1Stop-

apologise for this refuse, that originated from changes to the figures from an older version of the manuscript, as all the information concerning the in-vitro import into yeast mitochondria were contained in the Supplementary Methods, now moved to the main Materials and Methods. We have removed the comment about post translational modifications from the caption, as it more appropriately belongs in the discussion, were we hope it is now in the appropriate context: 'Once inside the mitochondria, it undergoes processing leading to the appearance of a lower molecular weight band, observed both in Western blots from whole cell and mice tissue protein extracts, as well as from purified mitochondria and in the yeast in vitro assays (Fig. 4C). The fragment is found predominantly in a carbonate- insoluble fraction, i.e. a membrane, and it appears to form a high molecular weight complex of ~180 kDa with other mitochondrial proteins (Fig. S7CD).'
. -Figure 4 Fig 4C in vitro import into yeast mitochondria: Legend does not seem correct.It is said line 428 « The kinetics pattern of M2tail is what it is expected for a protein imported in mitochondria: the appearance of the cleaved M2-tail is indicative of a post translational modifications of the fragment once imported in mitochondria.»I do not see the notion of post-translational modification here; what do the authors mean ?What is the line at 5% correspond to ?Nothing is indicated in the legend.We